6-Hydroxyequilenins as estrogenic agents

ABSTRACT

This invention provides estrogen receptor modulators of formula I, having the structure 
                         
wherein,
         R 1  is hydrogen, alkyl of 1–6 carbon atoms, benzyl, alkylcarbonyl of 2–7 carbon atoms, or benzoyl;   X is       
     
       
         
         
             
             
         
       
         
         
           
             R 2  is hydrogen, alkyl of 1–6 carbon atoms, benzyl, alkylcarbonyl of 2–7 carbon atoms, or benzoyl; 
             R 3  is hydrogen, alkyl of 1–6 carbon atoms, hydroxy, or alkoxy of 1–6 carbon atoms; 
             a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable salt of a sulfate ester of the hydroxyl group at the 3- or 17-position when R1 or R2 is hydrogen, or a glucuronide of the hydroxyl group at the 3- or 17-position when R1 or R2 is hydrogen.

This application claims priority from copending provisional applicationSer. No. 60/351,282, filed Jan. 23, 2002, the entire disclosure of whichis hereby incorporated by reference.

BACKGROUND OF THE INVENTION

This invention relates to 6-hydroxyequilenins, which are useful asestrogenic agents.

The pleiotropic effects of estrogens in mammalian tissues have been welldocumented, and it is now appreciated that estrogens affect many organsystems [Mendelsohn and Karas, New England Journal of Medicine 340:1801–1811 (1999), Epperson, et al., Psychosomatic Medicine 61: 676–697(1999), Crandall, Journal of Womens Health & Gender Based Medicine 8:1155–1166 (1999), Monk and Brodaty, Dementia & Geriatric CognitiveDisorders 11: 1–10 (2000), Hum and Macrae, Journal of Cerebral BloodFlow & Metabolism 20: 631–652 (2000), Calvin, Maturitas 34: 195–210(2000), Finking, et al., Zeitschrift fur Kardiologie 89: 442–453 (2000),Brincat, Maturitas 35: 107–117 (2000), Al-Azzawi, Postgraduate MedicalJournal 77: 292–304 (2001)]. Estrogens can exert effects on tissues inseveral ways, and the most well characterized mechanism of action istheir interaction with estrogen receptors leading to alterations in genetranscription. Estrogen receptors are ligand-activated transcriptionfactors and belong to the nuclear hormone receptor superfamily. Othermembers of this family include the progesterone, androgen,glucocorticoid and mineralocorticoid receptors. Upon binding ligand,these receptors dimerize and can activate gene transcription either bydirectly binding to specific sequences on DNA (known as responseelements) or by interacting with other transcription factors (such asAP1), which in turn bind directly to specific DNA sequences [Moggs andOrphamides, EMBO Reports 2: 775–781 (2001), Hall, et al., Journal ofBiological Chemistry 276: 36869–36872 (2001), McDonnell, Principles OfMolecular Regulation. p351–361(2000)]. A class of “coregulatory”proteins can also interact with the ligand-bound receptor and furthermodulate its transcriptional activity [McKenna, et al., EndocrineReviews 20: 321–344 (1999)]. It has also been shown that estrogenreceptors can suppress NFκB-mediated transcription in both aligand-dependent and independent manner [Quaedackers, et al.,Endocrinology 142: 1156–1166 (2001), Bhat, et al., Journal of SteroidBiochemistry & Molecular Biology 67: 233–240 (1998), Pelzer, et al.,Biochemical & Biophysical Research Communications 286: 1153–7 (2001)].

Estrogen receptors can also be activated by phosphorylation. Thisphosphorylation is mediated by growth factors such as EGF and causeschanges in gene transcription in the absence of ligand [Moggs andOrphamides, EMBO Reports 2: 775–781 (2001), Hall, et al., Journal ofBiological Chemistry 276: 36869–36872 (2001)].

A less well-characterized means by which estrogens can affect cells isthrough a so-called membrane receptor. The existence of such a receptoris controversial, but it has been well documented that estrogens canelicit very rapid non-genomic responses from cells. The molecular entityresponsible for transducing these effects has not been definitivelyisolated, but there is evidence to suggest it is at least related to thenuclear forms of the estrogen receptors [Levin, Journal of AppliedPhysiology 91: 1860–1867 (2001), Levin, Trends in Endocrinology &Metabolism 10: 374–377 (1999)].

Two estrogen receptors have been discovered to date. The first estrogenreceptor was cloned about 15 years ago and is now referred to as ERα[Green, et al., Nature 320: 134–9 (1986)]. The second form of theestrogen receptor was found comparatively recently and is called ERβ[Kuiper, et al., Proceedings of the National Academy of Sciences of theUnited States of America 93: 5925–5930 (1996)]. Early work on ERβfocused on defining its affinity for a variety of ligands and indeed,some differences with ERα were seen. The tissue distribution of ERβ hasbeen well mapped in the rodent and it is not coincident with ERα.Tissues such as the mouse and rat uterus express predominantly ERα,whereas the mouse and rat lung express predominantly ERβ [Couse, et al.,Endocrinology 138: 4613–4621 (1997), Kuiper, et al., Endocrinology 138:863–870 (1997)]. Even within the same organ, the distribution of ERα andERβ can be compartmentalized. For example, in the mouse ovary, ERβ ishighly expressed in the granulosa cells and ERα is restricted to thethecal and stromal cells [Sar and Welsch, Endocrinology 140: 963–971(1999), Fitzpatrick, et al., Endocrinology 140: 2581–2591 (1999)].However, there are examples where the receptors are coexpressed andthere is evidence from in vitro studies that ERα and ERβ can formheterodimers [Cowley, et al., Journal of Biological Chemistry 272:19858–19862 (1997)].

A large number of compounds have been described that either mimic orblock the activity of 17β-estradiol. Compounds having roughly the samebiological effects as 17β-estradiol, the most potent endogenousestrogen, are referred to as “estrogen receptor agonists”. Those which,when given in combination with 17β-estradiol, block its effects arecalled “estrogen receptor antagonists”. In reality there is a continuumbetween estrogen receptor agonist and estrogen receptor antagonistactivity and indeed some compounds behave as estrogen receptor agonistsin some tissues and estrogen receptor antagonists in others. Thesecompounds with mixed activity are called selective estrogen receptormodulators (SERMS) and are therapeutically useful agents (e.g. EVISTA)[McDonnell, Journal of the Society for Gynecologic Investigation 7:S10–S15 (2000), Goldstein, et al., Human Reproduction Update 6: 212–224(2000)]. The precise reason why the same compound can have cell-specificeffects has not been elucidated, but the differences in receptorconformation and/or in the milieu of coregulatory proteins have beensuggested.

It has been known for some time that estrogen receptors adopt differentconformations when binding ligands. However, the consequence andsubtlety of these changes has been only recently revealed. The threedimensional structures of ERα and ERβ have been solved byco-crystallization with various ligands and clearly show therepositioning of helix 12 in the presence of an estrogen receptorantagonist which sterically hinders the protein sequences required forreceptor-coregulatory protein interaction [Pike, et al., Embo 18:4608–4618 (1999), Shiau, et al., Cell 95: 927–937 (1998)]. In addition,the technique of phage display has been used to identify peptides thatinteract with estrogen receptors in the presence of different ligands[Paige, et al., Proceedings of the National Academy of Sciences of theUnited States of America 96: 3999–4004 (1999)]. For example, a peptidewas identified that distinguished between ERα bound to the full estrogenreceptor agonists 17β-estradiol and diethylstilbesterol. A differentpeptide was shown to distinguish between clomiphene bound to ERα andERβ. These data indicate that each ligand potentially places thereceptor in a unique and unpredictable conformation that is likely tohave distinct biological activities.

As mentioned above, estrogens affect a panoply of biological processes.In addition, where gender differences have been described (e.g. diseasefrequencies, responses to challenge, etc), it is possible that theexplanation involves the difference in estrogen levels between males andfemales.

DESCRIPTION OF THE INVENTION

This invention provides estrogenic compound of formula I having thestructure,

wherein,

-   R₁ is hydrogen, alkyl of 1–6 carbon atoms, benzyl, alkylcarbonyl of    2–7 carbon atoms, or benzoyl;    X is

-   R₂ is hydrogen, alkyl of 1–6 carbon atoms, benzyl, alkylcarbonyl of    2–7 carbon atoms, or benzoyl;-   R₃ is hydrogen, alkyl of 1–6 carbon atoms, hydroxy, or alkoxy of 1–6    carbon atoms;-   a pharmaceutically acceptable salt thereof, a pharmaceutically    acceptable salt of a sulfate ester of the hydrdoxyl group at the 3-    or 17-position when R₁ or R₂ is hydrogen, or a glucuronide of the    hydrdoxyl group at the 3- or 17-position when R₁ or R₂ is hydrogen.

Pharmaceutically acceptable salts can be formed from organic andinorganic bases, such as alkali metal salts (for example, sodium,lithium, or potassium) alkaline earth metal salts, ammonium salts,alkylammonium salts containing 1–6 carbon atoms or dialkylammonium saltscontaining 1–6 carbon atoms in each alkyl group, and trialkylammoniumsalts containing 1–6 carbon atoms in each alkyl group, when a compoundof this invention contains an acidic moiety, such as a free hydroxyl orsulfate ester group.

The term alkyl includes both branched and straight chain moieties.Examples include methyl, ethyl, propyl, butyl, isopropyl, sec-butyl,tert-butyl, and the like. The term alkylcarbonyl means an alkyl-C(O)—moiety, for example, acetyl.

As used in accordance with this invention, the term “providing,” withrespect to providing a compound or substance covered by this invention,means either directly administering such a compound or substance, oradministering a prodrug, derivative, or analog which will form theeffective amount of the compound or substance within the body.

Of the compounds of this invention, it is preferred that R₁ is hydrogenor a pharmaceutically acceptable salt of the sulfate ester at thathydroxyl group. It is also preferred that when X contains R₂, R₂ ishydrogen or a pharmaceutically acceptable salt of the sulfate ester atthat hydroxyl group, and R₃ is hydrogen. It is preferred that thepharmaceutically acceptable salt is sodium.

The reagents used in the preparation of the compounds of this inventioncan be either commercially obtained or can be prepared by standardprocedures described in the literature.

The compounds of the present invention can be prepared according to thefollowing synthetic scheme.

Referring to Scheme I, the common intermediate6-hydroxyequilenin-3,17β-diacetate (4) can be prepared from7-bromo-6-ketoestradiol-3–17β-diacetate (3) by elimination with CaCO3 inrefluxing dimethylacetamide [M. Harnik, Israel J. Chem, 1965, 3,183–192].

The acetyl protecting groups can be removed under trans esterificationconditions to provide the 6-hydroxyequilenin-17β-ol (5). The6-hydroxyequilenin (1) can be obtained by oxidation of compound 5 underDMSO, triethylaminesulfurtrioxide conditions (Shwarz., et al;Tetrahedron, 1994, 50,10709–10720).

Sodium-6-hydroxyequilenin-3-sulfate (2) can be synthesized from thecommon intermediate, 6-hydroxyequilenin-3,17β-diacetate (3) byprotecting the 6-hydroxy with a tert-butyldimethylsilyl group. The6-OTBDMS-equilenin-3,17β-diacetate (6) under trans esterificationconditions affords the 6-OTBDMS-equilenin-3,17β-diol (7). The 17-hydroxyfunction can be oxidized to 17-keto giving rise to 6-OTBDMS-equilenin(8). The latter (8) can be sulfated at the 3-hydroxy position to affordtriethylammonium-6-OTBDMS-equilenin-3-sulfate (9). Finally desilylationof compound 9 affords the sodium-6-hydroxyequilenin-3-sulfate (2).

Further derivitization at the 17-position (i.e., R₃ is not hydrogen) canbe accomplished via nucleophilic addition to the 17-carbonyl of compound(1).

Standard pharmacological test procedures are readily available todetermine the activity profile of a given test compound. The followingbriefly summarizes several representative test procedures and mayinclude data for representative compounds of the invention. All assays,except the radioligand binding assay, can be used to detect estrogenreceptor agonist or antagonist activity of compounds. In general,estrogen receptor agonist activity is measured by comparing the activityof the compound to a reference estrogen (e.g. 17β-estradiol,17α-ethinyl, 17β-estradiol, estrone, diethylstilbesterol etc). Estrogenreceptor antagonist activity is generally measured by co-treating thetest compound with the reference estrogen and comparing the result tothat obtained with the reference estrogen alone. Standardpharmacological test procedures for SERMs are also provided in U.S. Pat.Nos. 4,418,068 and 5,998,402 which are hereby incorporated by reference.

Evaluation of Binding Affinities to ERα and ERβ

Representative examples of the invention were evaluated for theirability to compete with 17β-estradiol for both ERα and ERβ in aconventional radioligand binding assay. This test procedure provides themethodology for one to determine the relative binding affinities for theERα or ERβ recptors. The procedure used is briefly described below.

Preparation of receptor extracts for characterization of bindingselectivity. The ligand binding domains, conveniently defined here asall sequence downstream of the DNA binding domain, were obtained by PCRusing full length cDNA as templates and primers that containedappropriate restriction sites for subcloning while maintaining theappropriate reading frame for expression. These templates containedamino acids M₂₅₀–V₅₉₅ of human ERα [Green, et al., Nature 320: 134–9(1986)] and M₂₁₄–Q₅₃₀ of human ERβ [Ogawa, et al., Biochemical &Biophysical Research Communications 243: 122–6 (1998)]. Human ERβ wascloned into pET15b (Novagen, Madison Wis.) as a Nco1-BamH1 fragmentbearing a C-terminal Flag tag. Human ERα was cloned as for human ERβexcept that an N-terminal His tag was added. The sequences of allconstructs used were verified by complete sequencing of both strands.

BL21(DE3) cells were used to express the human proteins. Typically a 10mL overnight culture was used to inoculate a 1 L culture of LB mediumcontaining 100 μg/mL of ampicillin. After incubation overnight at 37°C., IPTG was added to a final concentration of 1 mM and incubationproceeded at 25° C. for 2 hours. Cells were harvested by centrifugation(1500×g), and the pellets washed with and resuspended in 100 mL of 50 mMTris-Cl (pH 7.4), 150 mM NaCl. Cells were lysed by passing twice througha French press at 12000 psi. The lysate was clarified by centrifugationat 12,000×g for 30 minutes at 4° C. and stored at −70° C.

Evaluation of extracts for specific [³H]-estradiol binding. Dulbecco'sphosphate buffered saline (Gibco, 1×final concentration) supplementedwith 1 mM EDTA was used as the assay buffer. To optimize the amount ofreceptor to use in the assay, [³H]-17β-estradiol (New England Nuclear;final concentration=2 nM) ±0.6 μM diethlystilbestrol and 100 μL ofvarious dilutions of the E. coli lysate were added to each well of ahigh binding masked microtiter plate (EG&G Wallac). The final assayvolume was 120 μL and the concentration of DMSO was ≦1%. Afterincubation at room temperature for 5–18 hours, unbound material wasaspirated and the plate washed three times with approximately 300 μL ofassay buffer. After washing, 135 μL of scintillation cocktail (OptiphaseSupermix, EG&G Wallac) was added to the wells, and the plate was sealedand agitated for at least 5 minutes to mix scintillant with residualwash buffer. Bound radioactivity was evaluated by liquid scintillationcounting (EG&G Wallac Microbeta Plus).

After determining the dilution of each receptor preparation thatprovided maximum specific binding, the assay was further optimized byestimating the IC₅₀ of unlabelled 17β-estradiol using various dilutionsof the receptor preparation. A final working dilution for each receptorpreparation was chosen for which the IC₅₀ of unlabelled 17β-estradiolwas 2–4 nM.

Ligand binding competition test procedure. Test compounds were initiallysolubilized in DMSO and the final concentration of DMSO in the bindingassay was ≦1%. Eight dilutions of each test compound were used as anunlabelled competitor for [³H]-17β-estradiol. Typically, a set ofcompound dilutions would be tested simultaneously on human ERα and ERβ.The results were plotted as measured DPM vs. concentration of testcompound. For dose-response curve fitting, a four parameter logisticmodel on the transformed, weighted data was fit and the IC₅₀ was definedas the concentration of compound decreasing maximum [³H]-estradiolbinding by 50%.

Binding affinities for ERα and ERβ (as measured by IC₅₀) forrepresentative examples of the invention are shown in Table (1).

TABLE 1 Estrogen receptor binding affinities of compounds of theinvention Compound ER-β IC₅₀ (nM) ER-α IC₅₀ (nM) 1 123 2183 5 16 238

The results obtained in the standard pharmacologic test proceduredescribed above demonstrate that the compounds of this invention bindboth subtypes of the estrogen receptor. The IC₅₀s are generally lowerfor ERβ, indicating these compounds are preferentially ERβ selectiveligands, but are still considered active at ERα. Compounds of thisinvention will exhibit a range of activity based, at least partially, ontheir receptor affinity selectivity profiles. Since the compounds of theinvention bind ER-β with higher affinity than ER-α, they will be usefulin treating or inhibiting diseases that can be modulated via ER-β.Additionally, since each receptor ligand complex is unique and thus itsinteraction with various coregulatory proteins is unique, compounds ofthis invention will display different and unpredictable activitiesdepending on cellular context. For example, in some cell-types, it ispossible for a compound to behave as an estrogen receptor agonist whilein other tissues, an estrogen receptor antagonist. Compounds with suchactivity have sometimes been referred to as SERMs (Selective EstrogenReceptor Modulators). Unlike many estrogens, however, many of the SERMsdo not cause increases in uterine wet weight. These compounds areantiestrogenic in the uterus and can completely antagonize the trophiceffects of estrogen receptor agonists in uterine tissue. Thesecompounds, however, act as estrogen receptor agonists in the bone,cardiovascular, and central nervous systems. Due to this tissueselective nature of these compounds, they are useful in treating orinhibiting in a mammal disease states or syndromes which are caused orassociated with an estrogen deficiency (in certain tissues such as boneor cardiovascular) or an excess of estrogen (in the uterus or mammaryglands). In addition, compounds of this invention also have thepotential to behave as estrogen receptor agonists on one receptor typewhile behaving as estrogen receptor antagonists on the other. Forexample, it has been demonstrated that compounds can be antagonize theaction of 17β-estradiol via ERβ while exhibiting estrogen receptoragonist activity with ERα [Sun, et al., Endocrinology 140: 800–804(1999)]. Such ERSAA (Estrogen Receptor Selective Agonist Antagonist)activity provides for pharmacologically distinct estrogenic activitywithin this series of compounds

Regulation of Metallothionein II mRNA

Estrogens acting through ERβ, but not ERα can upregulate metallothioneinII mRNA levels in Saos-2 cells as described by Harris [Endocrinology142: 645–652 (2001)]. Results from this test procedure can be combinedwith results from the test procedure described below (ERE reporter testprocedure) to generate a selectivity profile for compounds of thisinvention (see also WO 00/37681). Data for representative compounds ofthe invention are shown in Table (2).

TABLE 2 Upregulation of metallothionein-II mRNA in Saos-2 cells byselected compounds of the invention Compound Fold upregulation 1 11 5 11Evaluation of Test Compound Using an ERE-Reporter Test Procedure inMCF-7 Breast Cancer Cells

Stock solutions of test compounds (usually 0.1 M) are prepared in DMSOand then diluted 10 to 100-fold with DMSO to make working solutions of 1or 10 mM. The DMSO stocks are stored at either 4° C. (0.1 M) or −20° C.(<0.1M). MCF-7 cells are passaged twice a week with growth medium[D-MEM/F-12 medium containing 10% (v/v) heat-inactivated fetal bovineserum, 1% (v/v) Penicillin-Streptomycin, and 2 mM glutaMax-1]. The cellsare maintained in vented flasks at 37° C. inside a 5% CO₂/95% humidifiedair incubator. One day prior to treatment, the cells are plated withgrowth medium at 25,000 cells/well into 96 well plates and incubated at37° C. overnight.

The cells are infected for 2 hr at 37° C. with 50 μl/well of a 1:10dilution of adenovirus 5-ERE-tk-luciferase in experimental medium[phenol red-free D-MEM/F-12 medium containing 10% (v/v) heat-inactivedcharcoal-stripped fetal bovine serum, 1% (v/v) Penicillin-Streptomycin,2 mM glutaMax-1, 1 mM sodium pyruvate]. The wells are then washed oncewith 150 μl of experimental medium. Finally, the cells are treated for24 hr at 37° C. in replicates of 8 wells/treatment with 150 μl/well ofvehicle (≦0.1% v/v DMSO) or compound that is diluted ≧1000-fold intoexperimental medium.

Initial screening of test compounds is done at a single dose of 1 μMthat is tested alone (estrogen receptor agonist mode) or in combinationwith 0.1 nM 17β-estradiol (EC₈₀; estrogen receptor antagonist mode).Each 96 well plate also includes a vehicle control group (0.1% v/v DMSO)and an estrogen receptor agonist control group (either 0.1 or 1 nM17β-estradiol). Dose-response experiments are performed in either theestrogen receptor agonist and/or estrogen receptor antagonist modes onactive compounds in log increases from 10⁻¹⁴ to 10⁻⁵ M. From thesedose-response curves, EC₅₀ and IC₅₀ values, respectively, are generated.The final well in each treatment group contains 5 μl of 3×10⁻⁵ MICI-182,780 (10⁻⁶ M final concentration) as an estrogen receptorantagonist control.

After treatment, the cells are lysed on a shaker for 15 min with 25μl/well of 1×cell culture lysis reagent (Promega Corporation). The celllysates (20 μl) are transferred to a 96 well luminometer plate, andluciferase activity is measured in a MicroLumat LB 96 P luminometer (EG& G Berthold) using 100 μl/well of luciferase substrate (PromegaCorporation). Prior to the injection of substrate, a 1 second backgroundmeasurement is made for each well. Following the injection of substrate,luciferase activity is measured for 10 seconds after a 1 second delay.The data are transferred from the luminometer to a Macintosh personalcomputer and analyzed using the JMP software (SAS Institute); thisprogram subtracts the background reading from the luciferase measurementfor each well and then determines the mean and standard deviation ofeach treatment.

The luciferase data are transformed by logarithms, and the HuberM-estimator is used to down-weight the outlying transformedobservations. The JMP software is used to analyze the transformed andweighted data for one-way ANOVA (Dunnett's test). The compoundtreatments are compared to the vehicle control results in the estrogenreceptor agonist mode, or the positive estrogen receptor agonist controlresults (0.1 nM 17β-estradiol) in the estrogen receptor antagonist mode.For the initial single dose experiment, if the compound treatmentresults are significantly different from the appropriate control(p<0.05), then the results are reported as the percent relative to the17β-estradiol control [i.e., ((compound−vehicle control)/(17β-estradiolcontrol−vehicle control))×100]. The JMP software is also used todetermine the EC₅₀ and/or IC₅₀ values from the non-linear dose-responsecurves.

Evaluation of Uterotrophic Activity

Uterotrophic activity of a test compound can be measured according tothe following standard pharmacological test procedures.

Procedure 1: Sexually immature (18 days of age) Sprague-Dawley rats areobtained from Taconic and provided unrestricted access to a casein-baseddiet (Purina Mills 5K96C) and water. On day 19, 20 and 21 the rats aredosed subcutaneously with 17α-ethinyl-17β-estradiol (0.06 μg/rat/day),test compound or vehicle (50% DMSO/50% Dulbecco's PBS). To assessestrogen receptor antagonist, compounds are coadministered with17α-ethinyl-17β-estradiol (0.06 μg/rat/day). There are six rats/groupand they are euthanized approximately 24 hours after the last injectionby CO₂ asphyxiation and pneumothorax. Uteri are removed and weighedafter trimming associated fat and expressing any internal fluid. Atissue sample can also be snap frozen for analysis of gene expression(e.g. complement factor 3 mRNA).

Procedure 2: Sexually immature (18 days of age) 129 SvE mice areobtained from Taconic and provided unrestricted access to a casein-baseddiet (Purina Mills 5K96C) and water. On day 22, 23, 24 and 25 the miceare dosed subcutaneously with compound or vehicle (corn oil). There aresix mice/group and they are euthanized approximately 6 hours after thelast injection by CO₂ asphyxiation and pneumothorax. Uteri are removedand weighed after trimming associated fat and expressing any internalfluid.

Evaluation of Osteoporosis and Lipid Modulation (Cardioprotection)

Female Sprague-Dawley rats, ovariectomized or sham operated, areobtained 1 day after surgery from Taconic Farms (weight range 240–275g). They are housed 3 or 4 rats/cage in a room on a 12/12 (light/dark)schedule and provided with food (Purina 5K96C rat chow) and water adlibitum. Treatment for all studies begin 1 day after arrival and ratsare dosed 7 days per week as indicated for 6 weeks. A group of agematched sham operated rats not receiving any treatment serve as anintact, estrogen replete control group for each study.

All test compounds are prepared in a vehicle of 50% DMSO (JT Baker,Phillipsburg, N.J.)/1×Dulbecco's phosphate saline (GibcoBRL, GrandIsland, N.Y.) at defined concentrations so that the treatment volume is0.1 mL/100 g body weight. 17β-estradiol is dissolved in corn oil (20μg/mL) and delivered subcutaneously, 0.1 murat. All dosages are adjustedat three week intervals according to group mean body weightmeasurements, and given subcutaneously.

Five weeks after the initiation of treatment and one week prior to thetermination of the study, each rat is evaluated for bone mineral density(BMD). The total and trabecular density of the proximal tibia areevaluated in anesthetized rats using an XCT-960M (pQCT; StratecMedizintechnik, Pforzheim, Germany). The measurements are performed asfollows: Fifteen minutes prior to scanning, each rat is anesthetizedwith an intraperitoneal injection of 45 mg/kg ketamine, 8.5 mg/kgxylazine, and 1.5 mg/kg acepromazine.

The right hind limb is passed through a polycarbonate tube with adiameter of 25 mm and taped to an acrylic frame with the ankle joint ata 90° angle and the knee joint at 180°. The polycarbonate tube isaffixed to a sliding platform that maintains it perpendicular to theaperture of the pQCT. The platform is adjusted so that the distal end ofthe femur and the proximal end of the tibia is in the scanning field. Atwo dimensional scout view is run for a length of 10 mm and a lineresolution of 0.2 mm. After the scout view is displayed on the monitor,the proximal end of the tibia is located. The PQCT scan is initiated 3.4mm distal from this point. The pQCT scan is 1 mm thick, has a voxel(three dimensional pixel) size of 0.140 mm, and consists of 145projections through the slice.

After the pQCT scan is completed, the image is displayed on the monitor.A region of interest including the tibia but excluding the fibula isoutlined. The soft tissue is mathematically removed using an iterativealgorithm. The density of the remaining bone (total density) is reportedin mg/cm³. The outer 55% of the bone is mathematically peeled away in aconcentric spiral. The density of the remaining bone (Trabeculardensity) is reported in mg/cm³.

One week after BMD evaluation the rats are euthanized by CO₂asphyxiation and pneumothorax, and blood is collected for cholesteroldetermination. The uteri are also removed and the weighed after trimmingassociated fat and expressing any luminal fluid. Total cholesterol isdetermined using a Boehringer-Mannheim Hitachi 911 clinical analyzerusing the Cholesterol/HP kit. Statistics were compared using one-wayanalysis of variance with Dunnet's test.

Evaluation of Antioxidant Activity

Porcine aortas are obtained from an abattoir, washed, transported inchilled PBS, and aortic endothelial cells are harvested. To harvest thecells, the intercostal vessels of the aorta are tied off and one end ofthe aorta clamped. Fresh, sterile filtered, 0.2% collagenase (Sigma TypeI) is placed in the vessel and the other end of the vessel then clampedto form a closed system. The aorta is incubated at 37° C. for 15–20minutes, after which the collagenase solution is collected andcentrifuged for 5 minutes at 2000×g. Each pellet is suspended in 7 mL ofendothelial cell culture medium consisting of phenol red free DMEM/Ham'sF12 media supplemented with charcoal stripped FBS (5%), NuSerum (5%),L-glutamine (4 mM), penicillin-streptomycin (1000 U/ml, 100 μg/ml) andgentamycin (75 μg/ml), seeded in 100 mm petri dish and incubated at 37°C. in 5%CO₂. After 20 minutes, the cells are rinsed with PBS and freshmedium added, this was repeated again at 24 hours. The cells areconfluent after approximately 1 week. The endothelial cells areroutinely fed twice a week and, when confluent, trypsinized and seededat a 1:7 ratio. Cell mediated oxidation of 12.5 μg/mL LDL is allowed toproceed in the presence of the compound to be evaluated (5 μM) for 4hours at 37° C. Results are expressed as the percent inhibition of theoxidative process as measured by the TBARS (thiobarbituric acid reactivesubstances) method for analysis of free aldehydes [Yagi, BiochemicalMedicine 15: 212–6 (1976)].

Progesterone Receptor mRNA Regulation Standard Pharmacological TestProcedure

This test procedure can be used to evaluate the estrogenic orantiestrogenic activity of compounds from this invention [Shughrue, etal., Endocrinology 138: 5476–5484 (1997)].

Rat Hot Flush Test Procedure

The effect of test compounds on hot flushes can be evaluated in astandard pharmacological test procedure which measures the ability of atest compound to blunt the increase in tail skin temperature whichoccurs as morphine-addicted rats are acutely withdrawn from the drugusing naloxone [Merchenthaler, et al., Maturitas 30: 307–16 (1998)]. Itcan also be used to detect estrogen receptor antagonist activity byco-dosing test compound with the reference estrogen.

Evaluation of Vasomotor Function in Isolated Rat Aortic Rings

Sprague-Dawley rats (240–260 grams) are divided into 4 groups:

-   1. Normal non-ovariectomized (intact)-   2. Ovariectomized (ovex) vehicle treated-   3. Ovariectomized 17β-estradiol treated (1 mg/kg/day)-   4. Ovariectomized animals treated with test compound (various doses)

Animals are ovariectomized approximately 3 weeks prior to treatment.Each animal receives either 17-β estradiol sulfate (1 mg/kg/day) or testcompound suspended in distilled, deionized water with 1% tween-80 bygastric gavage. Vehicle treated animals received an appropriate volumeof the vehicle used in the drug treated groups.

Animals are euthanized by CO₂ inhalation and exsanguination. Thoracicaortae are rapidly removed and placed in 37° C. physiological solutionwith the following composition (mM): NaCl (54.7), KCl (5.0), NaHCO₃(25.0), MgCl₂ 2H₂O (2.5), D-glucose (11.8) and CaCl₂ (0.2) gassed withCO₂—O₂, 95%/5% for a final pH of 7.4. The advantitia is removed from theouter surface and the vessel is cut into 2–3 mm wide rings. Rings aresuspended in a 10 mL tissue bath with one end attached to the bottom ofthe bath and the other to a force transducer. A resting tension of 1gram is placed on the rings. Rings are equilibrated for 1 hour, signalsare acquired and analyzed.

After equilibration, the rings are exposed to increasing concentrationsof phenylephrine (10⁻⁸ to 10⁻⁴ M) and the tension recorded. Baths arethen rinsed 3 times with fresh buffer. After washout, 200 mM L-NAME isadded to the tissue bath and equilibrated for 30 minutes. Thephenylephrine concentration response curve is then repeated.

Evaluation of Cardioprotective Activity

Apolipoprotein E-deficient C57/B1J (apo E KO) mice are obtained fromTaconic Farms. All animal procedures are performed under strictcompliance to IACUC guidelines. Ovariectomized female apo E KO mice, 4–7weeks of age, are housed in shoe-box cages and were allowed free accessto food and water. The animals are randomized by weight into groups(n=12–15 mice per group). The animals are dosed with test compounds orestrogen (17β-estradiol sulfate at 1 mg/kg/day) in the diet using aPrecise-dosing Protocol, where the amount of diet consumed is measuredweekly, and the dose adjusted accordingly, based on animal weight. Thediet used is a Western-style diet (57U5) that is prepared by Purina andcontains 0.50% cholesterol, 20% lard and 25 IU/KG Vitamin E. The animalsare dosed/fed using this paradigm for a period of 12 weeks. Controlanimals are fed the Western-style diet and receive no compound. At theend of the study period, the animals are euthanized and plasma samplesobtained. The hearts are perfused in situ, first with saline and thenwith neutral buffered 10% formalin solution.

For the determination of plasma lipids and lipoproteins, totalcholesterol and triglycerides are determined using enzymatic methodswith commercially available kits from Boehringer Mannheim and WakoBiochemicals, respectively and analyzed using the Boehringer MannheimHitachii 911 Analyzer. Separation and quantification of plasmalipoproteins were performed using FPLC size fractionation. Briefly,50–100 mL of serum is filtered and injected into Superose 12 andSuperose 6 columns connected in series and eluted at a constant flowrate with 1 mM sodium EDTA and 0.15 M NaCl. Areas of each curverepresenting VLDL, LDL and HDL are integrated using Waters Millennium™software, and each lipoprotein fraction is quantified by multiplying theTotal Cholesterol value by the relative percent area of each respectivechromatogram peak.

For the quantification of aortic atherosclerosis, the aortas arecarefully isolated and placed in formalin fixative for 48–72 hoursbefore handling. Atherosclerotic lesions are identified using Oil Red Ostaining. The vessels are briefly destained, and then imaged using aNikon SMU800 microscope fitted with a Sony 3CCD video camera system inconcert with IMAQ Configuration Utility (National Instrument) as theimage capturing software. The lesions are quantified en face along theaortic arch using a custom threshold utility software package (ColemanTechnologies). Automated lesion assessment is performed on the vesselsusing the threshold function of the program, specifically on the regioncontained within the aortic arch from the proximal edge of thebrachio-cephalic trunk to the distal edge of the left subclavian artery.Aortic atherosclerosis data are expressed as percent lesion involvementstrictly within this defined luminal area.

Evaluation of Cognition Enhancement

Ovariectomized rats (n=50) are habituated to an 8-arm radial arm mazefor 10-min periods on each of 5 consecutive days. Animals arewater-deprived prior to habituation and testing. A 100 μL aliquot ofwater placed at the ends of each arm serves as reinforcement.Acquisition of a win-shift task in the radial arm maze is accomplishedby allowing the animal to have access to one baited arm. After drinking,the animal exits the arm and re-enters the central compartment, where itnow has access to the previously visited arm or to a novel arm. Acorrect response is recorded when the animal chooses to enter a novelarm. Each animal is given 5 trials per day for 3 days. After the lastacquisition trial, the animals are assigned to one of the following 4groups:

1. Negative controls: injected with 10% DMSO/sesame oil vehicle oncedaily for 6 days (1 ml/kg, SC)

2. Positive controls: injected with 17β-estradiol benzoate for 2 daysand tested 4 days after the second injection (17β-estradiol benzoate at10 μg/0.1 mL per rat)

3. Estradiol: 17β-estradiol will be injected daily for 6 days (20 μg/kg,SC)

4. Test compound: injected daily for 6 days (doses vary).

All injections will begin after testing on the last day of acquisition.The last injection for groups 1, 3, and 4 will take place 2 hours beforetesting for working memory.

The test for working memory is a delayed non-matching-to-sample task(DNMS) utilizing delays of 15, 30, or 60 seconds. This task is avariation of the acquisition task in which the rat is placed in thecentral arena and allowed to enter one arm as before. A second arm isopened once the rat traverses halfway down the first arm, and again therat is required to choose this arm. When it has traveled halfway downthis second arm, both doors are closed and the delay is instituted. Oncethe delay has expired, both of the original two doors, and a third noveldoor, are opened simultaneously. A correct response is recorded when theanimal travels halfway down the third, novel arm. An incorrect responseis recorded when the animal travels halfway down either the first orsecond arms. Each animal will receive 5 trials at each of the threedelay intervals for a total of 15 trials per subject.

Evaluation of Effect on Pleurisy

The ability to reduce the symptoms of experimentally-induced pleurisy inrats can be evaluated according to the procedure of Cuzzocrea[Endocrinology 141: 1455–63 (2000)].

Evaluation of Protection Against Glutamate-Induced Cytotoxicity(Neuroprotection)

The neuroprotective activity of compounds of this invention can beevaluated in an in vitro standard pharmacological test procedure usingglutamate challenge [Zaulyanov, et al., Cellular & MolecularNeurobiology 19: 705–18 (1999); Prokai, et al., Journal of MedicinalChemistry 44: 110–4 (2001)].

Evaluation in the Mammary End Bud Test Procedure

Estrogens are required for full ductal elongation and branching of themammary ducts, and the subsequent development of lobulo-alveolar endbuds under the influence of progesterone. In this test procedure, themammotrophic activity of selected compounds of the invention can beevaluated according to the following standard pharmacological testprocedure. Twenty-eight day old Sprague-Dawley rats (Taconic Farms,Germantown, N.Y.) are ovariectomized and rested for nine days. Animalsare housed under a 12-hour light/dark cycle, fed a casein-based PurinaLaboratory Rodent Diet 5K96 (Purina, Richmond, Ind.) and allowed freeaccess to water. Rats were then dosed subcutaneously for six days withvehicle (50% DMSO (JT Baker, Phillipsburg, N.J.)/50% 1×Dulbecco'sPhosphate buffered saline (GibcoBRL, Grand Island, N.Y.), 17β-estradiol(0.1 mg/kg) or test compound (20 mg/kg). For the final three days, ratsare also dosed subcutaneously with progesterone (30 mg/kg). On theseventh day, rats are euthanised and a mammary fat pad excised. This fatpad is analyzed for casein kinase II mRNA as a marker of end budproliferation. Casein kinase II mRNA is anlayzed by real-time RT-PCR.Briefly, RNA is isolated following Trizol (GibcoBRL, Grand Island, N.Y.)according to the manufacture's directions, Samples are treated withDNAse I using DNA-free kit (Ambion), and casein kinase II mRNA levelsare measured by real-time RT-PCR using the Taqman Gold procedure (PEApplied Biosystems). A total of 50 ng of RNA is analyzed in triplicateusing casein kinase II specific primer pair (5′ primer,CACACGGATGGCGCATACT (SEQ.ID.NO: 1); 3′ primer, CTCGGGATGCACCATGAAG)(SEQ,ID.NO.: 2) and customized probe(TAMRA-CGGCACTGGTTTCCCTCACATGCT-FAM) (SEQ.ID.NO.: 3). Casein kinase IImRNA levels are normalized to 18s ribosomal RNA contained within eachsample reaction using primers and probe supplied by PE AppliedBiosystems.

Evaluation in the HLA Rat Standard Pharmacological Test Procedure forInflammatory Bowel Disease

Representative compounds can be evaluated in the HLA rat standardpharmacological test procedure which emulates inflammatory bowel diseasein humans. The following briefly describes the procedure used andresults obtained. Male HLA-B27 rats are obtained from Taconic andprovided unrestricted access to food (PMI Lab diet 5001) and water.Stool quality is observed daily and graded according to the followingscale: Diarrhea=3; soft stool=2; normal stool=1. At the end of thestudy, serum is collected and stored at −70° C. A section of colon isprepared for histological analysis and an additional segment is analyzedfor myeloperoxidase activity.

Histological analysis. Colonic tissue is immersed in 10% neutralbuffered formalin. Each specimen of colon is separated into four samplesfor evaluation. The formalin-fixed tissues are processed in a Tissue Tekvacuum infiltration processor (Miles, Inc; West Haven, Conn.) forparaffin embedding. The samples are sectioned at 5μm and then stainedwith hematoxylin and eosin (H&E) for blinded histologic evaluationsusing a scale modified after Boughton-Smith. After the scores arecompleted the samples are unblinded, and data are tabulated and analyzedby ANOVA linear modeling with multiple mean comparisons. Sections ofcolonic tissue are evaluated for several disease indicators and givenrelative scores.Evaluation in Two Models of Arthritis

Lewis rat assay of adjuvant-induced arthritis. Sixty, female, 12 weeksold, Lewis rats are housed according to standard facility operatingprocedures. They receive a standard regimen of food and water adlibitum. Each animal is identified by a cage card indicating the projectgroup and animal number. Each rat number is marked by indelible inkmarker on the tail. At least 10–21 days before study they areanesthetized and ovariectomized by standard aseptic surgical techniques.

Freund's Adjuvant-Complete (Sigma Immuno Chemicals, St. Louis, Mo.) isused to induce arthritis, each mL containing 1 mg Mycobacteriumtuberculosis heat killed and dried, 0.85 mL mineral oil and 0.15 mLmannide monooleate Lot No. 084H8800.

The following are examples of two test procedures. Inhibition testprocedure: Thirty rats are injected intradermally with 0.1 mL ofFreund's Adjuvant-Complete at the base of the tail. The animals arerandomized to four groups, each group containing six rats. Each day, thegroups receive vehicle (50% DMSO (JT Baker, Phillipsburg,N.J.)/1×Dulbecco's phosphate saline (GibcoBRL, Grand Island, N.Y.)) ortest compound (administered subcutaneously). All rats begin treatment onDay 1.

Treatment test procedure: Thirty rats are injected intradermally with0.1 mL of Freund's Adjuvant-Complete at the base of the tail. Theanimals are randomized to four groups, each group containing six rats.Each day, the groups receive vehicle (50% DMSO (J T Baker, Phillipsburg,N.J.)/1×Dulbecco's phosphate saline (GibcoBRL, Grand Island, N.Y.)) ortest compound (administered subcutaneously). All rats begin treatment onDay 8 after adjuvant injection.

Statistical analysis is performed using Abacus Concepts Super ANOVA.(Abacus Concepts, Inc., Berkeley, Calif.). All of the parameters ofinterest are subjected to Analysis of Variance with Duncan's newmultiple range post hoc testing between groups. Data are expressedthroughout as mean±standard deviation (SD), and differences are deemedsignificant if p<0.05.

The degree of arthritis severity is monitored daily in terms of thefollowing disease indices: Hindpaw erythema, hindpaw swelling,tenderness of the joints, and movements and posture. An integer scale of0 to 3 is used to quantify the level of erythema (0=normal paw, 1=milderythema, 2=moderate erythema, 3=severe erythema) and swelling (0=normalpaw, 1=mild swelling, 2=moderate swelling, 3=severe swelling of the hindpaw). The maximal score per day is 12.

At the end of the study the rats are euthanized with CO₂, hindlimbsremoved at necropsy and fixed in 10% buffered formalin, and the tarsaljoints decalcified and embedded in paraffin. Histologic sections arestained with Hematoxylin and Eosin or Saffranin O—Fast Green stain.

Slides are coded so that the examiner is blinded to the treatmentgroups. Synovial tissue from tarsal joints is evaluated based onsynovial hyperplasia, inflammatory cell infiltration, and pannusformation [Poole and Coombs, International Archives of Allergy & AppliedImmunology 54: 97–113 (1977)] as outlined below.

Category Grade 1. Synovial lining cells a. No change 0 b. Cellsenlarged, slightly thicknened 1 c. Cells enlarged, increase in numbers,moderately thickened. No 2 villus present d. Cells enlarged, thickened.Villus present 3 2. Fibroplasia a. No change 0 b. Fibroplasia presentunder lining cells 1 c. Small areas of areolar tissue replaced byfibrous tissue 2 d. Replacement of areloar tissue by fibrous tissue 3 3.Inflammatory cells a. Occasionally seen, scattered throughout selection0 b. Cells present in small numbers in or just under lining cell 1 layerand/or around blood vessels. c. Small focal collection of cells may bepresent 2 d. Large numbers of cells present in capsule and in or underlin- 3 ing cells layers. Large foci often seen. 4. Pannus a. Notdetectable 0 b. Detectable 1

In addition, articular cartilage and bone is evaluated using Mankin'shistological grading system [Mankin, et al., Journal of Bone & JointSurgery—American Volume 53: 523–37 (1971)] as shown below.

Category Grade 1. Structure a. Normal 0 b. Surface irregularly 1 c.Pannus and surface irregularity 2 d. Clefts to transitional zone 3 e.Clefts to radial zone 4 f. Clefts to calcified zone 5 g. Completedisorganization 6 2. Cells a. Normal 0 b. Diffuse hypercellularity 1 c.Cloning 2 d. Hypocellularity 3 3. Safranin-O staining a. Normal 0 b.Slight reduction 1 c. Modest reduction 2 d. Sever reduction 3 e. No dyenoted 4 4. Tidemark integrity a. Intact 0 b. Crossed by blood vessels 1

Evaluation in the HLA-B27 Rat model of arthritis. Representativecompounds are evaluated in the HLA-B27 rat standard pharmacological testprocedure which emulates arthritis in humans. The following brieflydescribes the procedure used. Male HLA-B27 rats are obtained fromTaconic and provided unrestricted access to a food (PMI Lab diet 5001)and water. Joint scores and histology are evaluated as described abovefor the Lewis rat model of adjuvant-induced arthritis.

Evaluation in in vivo Models of Carcinogeneisis

The ability of compounds of this invention to treat and inhibit variousmalignancies or hyperprolific disorders can be evaluated in standardpharmacological test procedures that are readily available in theliterature, and include the following two procedures.

Breast cancer. Athymic nu/nu (nude) mice are obtained ovariectomizedfrom Charles River Laboratories (Wilmington, Mass.). One day prior totumor cell injection, animals are implanted with time-release pelletscontaining 0.36–1.7 mg 17β-estradiol (60 or 90 day release, InnovativeResearch of America, Sarasota, Fla.) or a placebo. The pellet isintroduced subcutaneously into the intrascapular region using a 10-gaugeprecision trochar. Subsequently, mice are injected subcutaneously intothe breast tissue with either 1×10⁷ MCF-7 cells or 1×10⁷ BG-1 cells. Thecells are mixed with an equal volume of matrigel, a basement membranematrix preparation to enhance tumor establishment. Test compounds can beevaluated either by dosing one day after tumor cell implantation(inhibition regimen) or after tumors have reached a certain size(treatment regimen). Compounds are administered either intraperitoneallyor orally in a vehicle of 1% tween-80 in saline each day. Tumor size isevaluated every three or seven days.

Colon cancer. The ability to treat or inhibit colon cancer can beevaluated in the test procedure of Smirnoff [Oncology Research 11:255–64 (1999)].

Evaluation of Neuroprotection in Two in vivo Test Procedures

Transient global ischemia in the Mongolian gerbil. The effect of testcompounds on preventing or treating brain injury in response to oxygendeprivation/reperfusion can be measured using the following testprocedure.

Female Mongolian gerbils (60–80 g; Charles River Laboratories, Kingston,N.Y.) are housed in the Wyeth-Ayerst animal care facility (AAALACcertified) with a 12-hour light, 12-hour dark photoperiod and freeaccess to tap water and a low-estrogen casein diet (Purina; Richmond,Ind.). After acclimation (3–5 days), gerbils are anesthetized withisoflurane (2–3% mixture with O₂), ovariectomized (Day 0). Beginning thefollowing morning (Day 1), gerbils are treated subcutaneously each daywith either vehicle (10% ETOH/corn oil), 17β-estradiol (1 mg/kg, sc) oran experimental compound. On Day 6, gerbils (n=4–5/group) areanesthetized with isoflurane, the common carotid arteries visualized viaa mid-line neck incision and both arteries simultaneously occluded for 5minutes with non-traumatic micro aneurysm clips. After occlusion, theclips are removed to allow cerebral reperfusion and the neck incisionclosed with wound clips. All animals are fasted overnight prior to theglobal ischemia surgery, a step that facilitates consistent ischemicinjury. On Day 12, gerbils are exposed to a lethal dose of CO₂, and thebrains frozen on dry ice and stored at −80° C.

The degree of neuronal protection is evaluated by in situ hybridizationanalysis of neurogranin mRNA. Briefly, 20 μm coronal cryostat sectionsare collected on gelatin-coated slides, dried and stored at −80° C. Atthe time of processing, the desiccated slide boxes are warmed to roomtemperature, the slides postfixed in 4% paraformaldehyde, treated withacetic anhydride and then delipidated and dehydrated with chloroform andethanol. Processed section-mounted slides are then hybridized with 200μl (6×10⁶ DPM/slide) of an antisense or sense (control) riboprobe forNeurogranin (³⁵S-UTP-labeled NG-241; bases 99–340). in a 50% formamidehybridization mix and incubated overnight at 55° C. in a humidifiedslide chamber without coverslipping. The following morning, the slidesare collected in racks, immersed in 2×SSC (0.3 M NaCl, 0.03 M sodiumcitrate; pH 7.0)/10 mM DTT, treated with RNase A (20 μg/ml) and washed(2×30 min) at 67° C. in 0.1×SSC to remove nonspecific label. Afterdehydration, the slides are opposed to BioMax (BMR-1; Kodak) X-ray filmovernight.

The level of neurogranin hybridization signal is used to quantitativelyassess the degree of neuronal loss in the CA1 region after injury and toevaluate the efficacy of 17β-estradiol and experimental compounds.Neurogranin mRNA is selected for these studies because it is highlyexpressed in the hippocampal neurons including CA1, but absent in gliaand other cell types present in this brain region. Therefore,measurement of the amount of neurogranin mRNA present representssurviving neurons. Relative optical density measurements of neurograninhybridization signal are obtained from film autoradiograms with acomputer based image analysis system (C-Imaging Inc., Pittsburgh, Pa.).The results from 6 sections (40 μm apart) per animal are averaged andstatistically evaluated. Numerical values are reported as the mean±SEM.One-way analysis of variance is used to test for differences in thelevel of neurogranin mRNA and all statements of non-difference in theresults section imply that p>0.05.

Middle cerebral artery occlusion in mice. Neuroprotection can beevaluated according to the test procedures described by Dubal [see,Dubal, et al., Proceedings of the National Academy of Sciences of theUnited States of America 98: 1952–1957 (2001), Dubal, et al., Journal ofNeuroscience 19: 6385–6393 (1999)].

Ovulation Inhibition Standard Pharmacological Test Procedure

The test procedure is used to determine whether test compounds caninhibit or change the timing of ovulation. It can also be used todetermine the number of oocytes ovulated [Lundeen, et al., J SteroidBiochem Mol Biol 78: 137–143 (2001)].

Based on the results obtained in the standard pharmacological testprocedures, the compounds of this invention are estrogen receptormodulators useful in the treatment or inhibition of conditions,disorders, or disease states that are at least partially mediated by anestrogen deficiency or excess, or which may be treated or inhibitedthrough the use of an estrogenic agent. The compounds of this inventionare particularly useful in treating a peri-menopausal, menopausal, orpostmenopausal patient in which the levels of endogenous estrogensproduced are greatly diminished. Menopause is generally defined as thelast natural menstrual period and is characterized by the cessation ofovarian function, leading to the substantial diminution of circulatingestrogen in the bloodstream. As used herein, menopause also includesconditions of decreased estrogen production that may be surgically,chemically, or be caused by a disease state which leads to prematurediminution or cessation of ovarian function.

Accordingly, the compounds of this invention are useful in treating orinhibiting osteoporosis and in the inhibition of bone demineralization,which may result from an imbalance in a individual's formation of newbone tissues and the resorption of older tissues, leading to a net lossof bone. Such bone depletion results in a range of individuals,particularly in post-menopausal women, women who have undergonebilateral oophorectomy, those receiving or who have received extendedcorticosteroid therapies, those experiencing gonadal dysgenesis, andthose suffering from Cushing's syndrome. Special needs for bone,including teeth and oral bone, replacement can also be addressed usingthese compounds in individuals with bone fractures, defective bonestructures, and those receiving bone-related surgeries and/or theimplantation of prosthesis. In addition to those problems describedabove, these compounds can be used in treatment or inhibition forosteoarthritis, hypocalcemia, hypercalcemia, Paget's disease,osteomalacia, osteohalisteresis, multiple myeloma and other forms ofcancer having deleterious effects on bone tissues.

The compounds of this invention are also useful in inhibiting ortreating other effects of estrogen deprivation including, hot flushes,vaginal or vulvar atrophy, atrophic vaginitis, vaginal dryness,pruritus, dyspareunia, dysuria, frequent urination, urinaryincontinence, urinary tract infections. Other reproductive tract usesinclude the treatment or inhibition of dysfunctional uterine bleeding.

The compounds of this invention are also active in the brain and aretherefore useful for inhibiting or treating Alzheimer's disease,cognitive decline, decreased libido, senile dementia, neurodegenerativedisorders, depression, anxiety, insomnia, schizophrenia, andinfertility. The compounds of this invention are also useful in treatingor inhibiting benign or malignant abnormal tissue growth including,glomerulosclerosis, prostatic hypertrophy, uterine leiomyomas, breastcancer, scleroderma, fibromatosis, endometriosis, endometrial cancer,polycystic ovary syndrome, endometrial polyps, benign breast disease,adenomyosis, ovarian cancer, melanoma, prostate cancer, cancers of thecolon, CNS cancers, such as glioma or astioblastomia.

The compounds of this invention are cardioprotective and areantioxidants, and are useful in lowering cholesterol, triglycerides,Lp(a), and LDL levels; inhibiting or treating hypercholesteremia,hyperlipidemia, cardiovascular disease, atherosclerosis, peripheralvascular disease, restenosis, and vasospasm, and inhibiting vascularwall damage from cellular events leading toward immune mediated vasculardamage. The compounds of this invention are also useful in treatingdisorders associated with inflammation or autoimmune diseases, includinginflammatory bowel disease (Crohn's disease, ulcerative colitis,indeterminate colitis), arthritis (rheumatoid arthritis,spondyloarthropathies, osteoarthritis), pleurisy, ischemia/reperfusioninjury (e.g. stroke, transplant rejection, myocardial infarction, etc.),asthma, giant cell arteritis, prostatitis, uveitis, psoriasis, multiplesclerosis, systemic lupus erythematosus and sepsis.

The compounds of this invention are also useful in treating orinhibiting ocular disorders including cataracts, uveitis, and maculardegeneration and in treating skin conditions such as aging, alopecia,and acne.

The compounds of this invention are also useful in treating orinhibiting metabolic disorders such as type-II diabetes, of lipidmetabolism, appetite (e.g. anorexia nervosa and bulimia).

Compounds in this invention are also useful in treating or inhibitingbleeding disorders such as hereditary hemorrhagic telangiectasia,dysfunctional uterine bleeding, and combating hemorrhagic shock.

The compounds of this invention are useful in disease states whereamenorrhea is advantageous, such as leukemia, endometrial ablations,chronic renal or hepatic disease or coagulation diseases or disorders.

The compounds of this invention can be used as a contraceptive agent,particularly when combined with a progestin.

When administered for the treatment or inhibition of a particulardisease state or disorder, it is understood that the effective dosagemay vary depending upon the particular compound utilized, the mode ofadministration, the condition, and severity thereof, of the conditionbeing treated, as well as the various physical factors related to theindividual being treated. Effective administration of the compounds ofthis invention may be given at an oral dose of from about 0.1 mg/day toabout 1,000 mg/day. Preferably, administration will be from about 10mg/day to about 600 mg/day, more preferably from about 50 mg/day toabout 600 mg/day, in a single dose or in two or more divided doses. Theprojected daily dosages are expected to vary with route ofadministration.

Such doses may be administered in any manner useful in directing theactive compounds herein to the recipient's bloodstream, includingorally, via implants, parentally (including intravenous,intraperitoneal, intraarticularly and subcutaneous injections),rectally, intranasally, topically, ocularly (via eye drops), vaginally,and transdermally.

Oral formulations containing the active compounds of this invention maycomprise any conventionally used oral forms, including tablets,capsules, buccal forms, troches, lozenges and oral liquids, suspensionsor solutions. Capsules may contain mixtures of the active compound(s)with inert fillers and/or diluents such as the pharmaceuticallyacceptable starches (e.g. corn, potato or tapioca starch), sugars,artificial sweetening agents, powdered celluloses, such as crystallineand microcrystalline celluloses, flours, gelatins, gums, etc. Usefultablet formulations may be made by conventional compression, wetgranulation or dry granulation methods and utilize pharmaceuticallyacceptable diluents, binding agents, lubricants, disintegrants, surfacemodifying agents (including surfactants), suspending or stabilizingagents, including, but not limited to, magnesium stearate, stearic acid,talc, sodium lauryl sulfate, microcrystalline cellulose,carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginicacid, acacia gum, xanthan gum, sodium citrate, complex silicates,calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalciumphosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride,talc, dry starches and powdered sugar. Preferred surface modifyingagents include nonionic and anionic surface modifying agents.Representative examples of surface modifying agents include, but are notlimited to, poloxamer 188, benzalkonium chloride, calcium stearate,cetostearl alcohol, cetomacrogol emulsifying wax, sorbitan esters,colloidol silicon dioxide, phosphates, sodium dodecylsulfate, magnesiumaluminum silicate, and triethanolamine. Oral formulations herein mayutilize standard delay or time release formulations to alter theabsorption of the active compound(s). The oral formulation may alsoconsist of administering the active ingredient in water or a fruitjuice, containing appropriate solubilizers or emulsifiers as needed.

In some cases it may be desirable to administer the compounds directlyto the airways in the form of an aerosol.

The compounds of this invention may also be administered parenterally orintraperitoneally. Solutions or suspensions of these active compounds asa free base or pharmacologically acceptable salt can be prepared inwater suitably mixed with a surfactant such as hydroxy-propylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols and mixtures thereof in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to inhibitthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

For the purposes of this disclosure, transdermal administrations areunderstood to include all administrations across the surface of the bodyand the inner linings of bodily passages including epithelial andmucosal tissues. Such administrations may be carried out using thepresent compounds, or pharmaceutically acceptable salts thereof, inlotions, creams, foams, patches, suspensions, solutions, andsuppositories (rectal and vaginal).

Transdermal administration may be accomplished through the use of atransdermal patch containing the active compound and a carrier that isinert to the active compound, is non toxic to the skin, and allowsdelivery of the agent for systemic absorption into the blood stream viathe skin. The carrier may take any number of forms such as creams andointments, pastes, gels, and occlusive devices. The creams and ointmentsmay be viscous liquid or semisolid emulsions of either the oil-in-wateror water-in-oil type. Pastes comprised of absorptive powders dispersedin petroleum or hydrophilic petroleum containing the active ingredientmay also be suitable. A variety of occlusive devices may be used torelease the active ingredient into the blood stream such as asemi-permeable membrane covering a reservoir containing the activeingredient with or without a carrier, or a matrix containing the activeingredient. Other occlusive devices are known in the literature.

Suppository formulations may be made from traditional materials,including cocoa butter, with or without the addition of waxes to alterthe suppository's melting point, and glycerin. Water soluble suppositorybases, such as polyethylene glycols of various molecular weights, mayalso be used.

The preparation of representative examples of this invention isdescribed below.

EXAMPLE A

6-Hydroxy-3,17β-diacetoxydihydroequilenin (4)

Ref: M. Harnik, Israel Journal of Chemistry, Vol.3, 1965, p. 183–192.

A mixture of 6-keto-7-bromo-17β-estradiol-diacetate (3, 3.0 g, 0.007mol) and CaCO₃ (3.3 g, 0.033 mol) in dimethylacetamide (50 mL) wasrefluxed for 2 h. TLC (EtOAC-hexanes 4:6) showed no starting material,mostly the desired product. The reaction mixture was cooled to 25° C.,concentrated in vacuum to dryness, and the residue treated with EtOAc(150 mL) and 0.1 N HCl (75 mL). The aqueous layer was extracted withEtOAc (50 mL), and combined organic layer and extracts were washed withwater, saturated NaHCO₃, and brine, dried over Na₂SO₄, and concentratedin vacuum to dryness to give 4 (1.5 g, 62.5%) as a light brown oil.

-   ¹H NMR (CDCl₃): 7.91 (d, 1H, J=10.2 Hz); 7.86 (d, 1H, J=2.7 Hz);    7.26 (dd, 1H, J=2.8, 10.1 Hz); 6.56 (s, 1H); 5.07 (m, 1H); 4.88 (m,    1H, 17□—H); 3.25–3.08 (m, 2H); 2.87 (m, 1H); 2.46–1.73 (m, 6H); 2.36    (s, 3H, —OAc); 2.10 (s, 3H; —OAc); 0.73 (s, 3H, —CH₃). GC/MS: MW    368.

EXAMPLE B

6-Hydroxy-3,17β-dihydroequilenin (5)

To a solution of 4 (5.0 g, 0.014 mol) in methanol (50 mL), K₂CO₃ (3.75g, 0.027 mol) was added at 25° C., and the slurry was stirred for 18 h.Reaction mixture turned dark pink. TLC (EtOAC-hexanes 4:6) showed nostarting material, mostly the desired product. The reaction mixture wasconcentrated in vacuum to dryness, and the residue was chromatographedon Silica Gel column using gradient eluation (hexanes-EtOAc, 100:0 to50:50) to give 5 (3.3 g, 85.5%) as a colorless oil.

-   ¹H NMR (DMSO): 9.48 (s, 1H; —OH); 9.41 (s, 1H; —OH); 7.69 (d, 1H,    J=10.1 Hz); 7.34 (d, 1H, J=2.6 Hz); 7.02 (dd, 1H, J=2.8, 10.00 Hz);    6.48 (s, 1H); 4.64 (d, 1H, J=5.3 Hz; 17□—OH); 3.71 (m, 1H; 17□—H);    3.09–1.54 (m, 9H); 0.55 (s, 3H, —CH₃). GC/MS: MW 282.

EXAMPLE C

6-Hydroxyequilenin (1)

Ref: Shwarz S., et al, Tetrahedron, 50(36), 10709–10720(1994) To asolution of 5 (2.5 g, 0.009 mol) in triethylamine (11 mL, 0.080 mol) andDMSO (20 mL), sulfur trioxide trimethylamine complex (5.6 g, 0.040 mol)was added at room temperature with stirring. The mixture was stirred for3 h at 25° C., then diluted with water (150 mL), and extracted withEtOAc (3×50 mL). The combined organic extracts were washed with 0.1 NHCl and water, dried over Na₂SO₄, and concentrated in vacuum to drynessto afford 1 (2.3 g) as oily solid. The crude product was recrystallizedfrom iPrOH-toluene to give 1 (1.3 g, 51.5%) as a white solid.

-   ¹H NMR (DMSO): 9.65 (s, 1H, —OH); 9.50 (s, 1H; —OH); 7.71 (d, 1H,    J=10.1 Hz); 7.40 (d, 1H, J=2.8 Hz); 7.05 (dd, 1H, J=28.6, 10.1 Hz);    6.62 (s, 1H); 3.08–1.71 (m, 9H); 0.69 (s, 1H; —CH₃). GC/MS: MW 426.

EXAMPLE D

6-OTBDMS equilenin-3,17β-diacetate (6)

A mixture of 6-hydroxyequilenin-3, 17β-diacetate (4, 5.0 g, 13.58 mmol),tert. butyldimethylsilyl chloride (3.07 g, 20 mmol) and imidazole (2.78g, 40 mmol) in dimethylformamide (40 ml) was stirred at ambienttemperature under N₂ atmosphere. After 1 hour, the reaction mixture waspoured onto an ice-cold saturated sodium bicarbonate solution (200 ml).It was extracted with ethyl acetate (40 ml×3). The combined ethylacetate extracts were washed with water (100 ml) and brine (100 ml),dried (Na₂SO₄) and evaporated to give 6.2 g of the crude product. Thecrude product was purified by column chromatography (hexane:ethylacetate 8.5:1.5). Upon evaporation of the appropriate fractions, product6 (4.96 g, 76%) was obtained as a white solid.

-   ¹H-NMR (300 MHz, CDCl3): δ 7.90 (d, J=9.1 Hz, 1H), 7.83 (d, J=2.5    Hz, 1H), 7.23 (dd, J=9.1, 2.5, 1H), 6.62 (s, 1H), 4.88 (dd, J=9.1,    6.9, 1H), 3.0–3.3 (m, 2H), 2.88 (m, 1H), 2.44 (m, 1H), 2.36 (s, 3H),    2.17 (m, 2H), 2.1 (s, 3H), 1.7–1.89 (m, 3H), 1.07 (s, 9H), 0.74 (s,    3H), 0.29 (s, 3H), 0.26 (s, 3H). GC-MS: 482.-   HPLC (purity area %): 99.34%.

EXAMPLE E

6-OTBDMS equilenin-17β-ol (7)

T a solution of the diacetate 6 (0.35 g, 0.72 mmol) in methanol (5 ml)was added anhydrous potassium carbonate (0.1 g, 0.72 mmol). The reactionmixture was stirred at ambient temperature for 22 h. The reactionmixture was evaporated. To the residue, water (25 ml) was added andneutralized with 1 M HCl. The aqueous layer was extracted with ethylacetate (20 ml×3), washed with water (30 ml), brine (30 ml), dried andevaporated to give 0.29 g of the crude product 7. Silica gel columnchromatography of the crude product (hexane:ethyl acetate 7.5:2.5) gavepure white solid product 7 (0.194 g, 68%).

-   ¹H-NMR (300 MHz, CDCl3): δ 7.80 (d, J=9.1 Hz), 7.48 (d, J=2.7 Hz),    7.11 (dd, J=9.1, 2.7, 1H), 6.56 (s, 1H), 5.75 (brs, 1H), 3.96 (m,    1H), 3.04–3.26 (m, 2H), 2.78 (m, 1H), 2.37 (m, 1H), 2.08–2.26 (m,    2H), 1.6–1.82 (m, 4H), 1.07 (s, 9H), 0.71 (s, 3H), 0.29 (s, 3H),    0.26 (s, 3H).-   HPLC (purity area %): 99.8%.

EXAMPLE F

6-OTBDMS Equilenin (8)

To a solution of the diol 7 (1.3 g, 3.26 mmol) in dimethyl sulfoxide (10ml) at ambient temperature was added Et3N (0.99 g, 9.78 mmol) andEt₃N—SO₃ (1.47 g, 8.1 mmol). After 30 min, the pink reaction mixture waspoured into water (100 ml), stirred for 5 min. The aqueous solution wasextracted with ethyl acetate (24 ml×4), washed with water (25 ml×2),brine (50 ml×2), dried and evaporated to give purple colored solid crudeproduct. Silica gel column chromatography (hexane:ethyl acetate 21:4) ofthe crude product gave light pink colored solid product 8 (0.88 g, 68%).

-   ¹H-NMR (300 MHz, DMSO-d₆): δ 9.64 (s, 1 h), 7.74 (d, J=9.1 Hz, 1H),    7.34 (d, J=2.5 Hz, 1H), 7.06 (dd, J=9.1, 2.5 Hz), 6.59 (s, 1H),    2.94–3.19 (m, 3H), 2.61 (m, 1H), 2.23–2.46 (m, 2H), 1.98 (m, 1H),    1.68–1.91 (m, 2H), 1.04 (s, 9H), 0.67 (s, 3H), 0.26 (s, 3H), 0.23    (s, 3H).-   GC-MS: 99.6%-   HPLC (purity area %): 97.7%.

EXAMPLE G

Triethylammonium-6-OTBDMS-equilenin-3-sulfate Stablized with TRIS (9)

To a solution of 8 (0.81 g, 2.04 mmol) in anhydrous THF (50 ml) wasadded triethylamine sulfur trioxide complex (0.64 g, 3.48 mmol) at 22°C. Stirred for 20 h at 22° C., then the reaction mixture was evaporated.To the residue, a solution of tris(hydroxymethyl)aminomethane (TRIS,0.55 g) in water (150 ml) was added. The hazy solution was extractedwith diethyl ether (100 ml×3). The aqueous layer (200 ml) wasconcentrated (180 ml) on a rotovap and the resulting solution waslyophilized to give 9 stablized with TRIS, as a tan colored solid (1.65g, 76%).

-   ¹H-NMR (300 MHz, DMSO-d₆): δ 7.83 (m, 2H), 7.38 (dd, J=9.1, 2.4 Hz),    6.65 (s, 1H), 3.01–3.26 (m, 3H), 2.75 (q, J=7.2 Hz, 6H), 2.61 (m,    1H), 2.23–2.47 (m, 2H), 1.90 (m, 1H), 1.71–1.94 (m, 2H), 1.03 (m,    18H), 0.65 (s, 3H), 0.23 (s, 3H), 0.21 (s, 3H).-   HPLC (purity area %): 95.7%.

EXAMPLE H

Sodium-6-hydroxyequilenin-3-sulfate (2)

Step A

Sodium-6-OTBDMS Equilenin-3-Sulfate (10)

Anhydrous THF (40 mL) was delivered by cannula into a 100-mL 3-neckedround bottomed flask containing TBDMS protected 6-hydroxyequilenin 8(0.888 g, 2.24 mmole). The triethylamine sulfur trioxide complex (0.822g, 0.049 mole) was then added under a nitrogen atmosphere. The reactionmixture was stirred for 2¼ hr at 20 to 25° C. Analysis by LC/MSindicated 98.6% of the expected sulfate and 1.4% unreacted 8. The THFwas evaporated under reduced pressure (25 to 35 mm Hg) at 35° C.Distilled water (35 mL) was added along with sodium carbonate (0.88 g,8.3 mmole). The gummy aqueous mixture was extracted with 15 mL of ethylacetate. The aqueous phase containing the sulfate 10 was sampled andanalyzed by LC/MS. The mass spectrum (positive mode) of the major peakshowed three fragments: M/Z 477, M+1; 397, M-SO₃; 398, M-SO₃+1; and 419,M-SO₃+Na. A similar fragmentation pattern was observed in the negativemode. UV scans across the major peak were identical indicating the peakwas homogeneous. The aqueous solution of 10 was used in the followingstep without further purification.

Step B

Sodium-6-Hydroxyequilenin-3-Sulfate (2)

MeOH (100 mL) was added to the aqueous mixture of the sulfate ester (seeabove). Anhydrous sodium fluoride (0.206 g) was added to the reactionmixture. After 4.5 hr stirring at 25° C., LC/MS analysis indicated 53%of the de-silylated sulfate ester 2 and 36% unreacted 10. After stirringfor a total of 18 hr at 20 to 25° C., analysis by LC/MS 85% of thede-silylated sulfate 2, 4.8% of the starting material 10 and 0.7% of the3-hydroxy-6-TBDMS 8. The MeOH was removed by evaporation under reducedpressure (25 to 35-mm Hg, 40° C.). Distilled water (100 mL) was added tothe residue and the aqueous solution was extracted with diethyl ether(3×100 mL). Analysis of the aqueous phase by LC/MS indicated 90.1% ofthe 6-hydroxy-3-sulfate 2 (ret. time 3.30 min) and two minor impurities:3.88 min (1.4%) and 4.40 min (4.9%). The crude material was dissolved inanhydrous ethanol (50 mL) and filtered to remove a small amount ofinsolubles. LC/MS analysis of the filtrate indicated the 94.4% of 2 and2.0% and 3.2% of the other impurities (ret. times 3.6 and 4.0 minrespectively). Evaporation of the solvent (25 to 35 mm Hg, 40° C.)afforded 0.672 g of 2 (78% yield based on the sulfated TBDMS ether 7).FTIR (KBr) of the product was consistent with the expected structure 2with strong bands at 1726, 1640, 1413, 1260, 1054, 800 and 644 cm⁻¹. Theproduct 2 was combined with 0.448 g of tris(hydroxymethyl)aminomethane(TRIS, as stabilizer) in 100 mL of distilled water and lyophilized to afluffy tan powder (1.107 g).

-   ¹H-NMR (300 MHz, DMSO-d₆): δ 9.82 (s, 1H), 7.89 (d, J=2.0 Hz, 1H),    7.77 (d, J=7.0 Hz, 1H), 7.33 (dd, J=7.0, 2.0 Hz), 6.69 (s, 1H),    3.0–3.2 (m, 3H), 2.65 (m, 1H), 2.27–2.43 (m, 2H), 1.87–2.1 (m, 2H),    1.73–1.83 (m, 1H), 0.70 (s, 3H).-   FTIR (KBr): 1726, 1640, 1413, 1260, 1054, 800 and 644 cm⁻¹.-   LC/MS: 95%.

1. A method of treating or inhibiting inflammatory bowel disease,Crohn's disease, ulcerative proctitis, or colitis in a mammal in needthereof, which comprises administering to said mammal an effectiveamount of a compound of formula I, having the structure

wherein, R₁ is hydrogen, alkyl of 1–6 carbon atoms, benzyl,alkylcarbonyl of 2–7 carbon atoms, or benzoyl; X is

R₂ is hydrogen, alkyl of 1–6 carbon atoms, benzyl, alkylcarbonyl of 2–7carbon atoms, or benzoyl; R₃ is hydrogen, alkyl of 1–6 carbon atoms,hydroxy, or alkoxy of 1–6 carbon atoms; a pharmaceutically acceptablesalt thereof, a pharmaceutically acceptable salt of a sulfate ester ofthe hydroxyl group at the 3- or 17-position when R₁ or R₂ is hydrogen,or a glucuronide of the hydroxyl group at the 3- or 17-position when R₁or R₂ is hydrogen.
 2. The method according to claim 1, wherein thecompound provided is 6-hydroxy-3,17 β-dihydroequilenin, or apharmaceutically acceptable salt thereof, a pharmaceutically acceptablesalt of a sulfate thereof, or a glucuronide thereof.
 3. The methodaccording to claim 1, wherein the compound provided is6-hydroxyequilenin, or a pharmaceutically acceptable salt thereof, apharmaceutically acceptable salt of a sulfate thereof, or a glucuronidethereof.